The present invention relates to polymerase chain reaction (PCR) assays for quantification of DNA. More specifically, the invention relates to assays for the real-time quantification of human mitochondrial DNA (mtDNA).
Sequence analysis of human mtDNA has become a valuable forensic tool for specimens, such as hair and calcified tissue samples, in which nuclear DNA analysis may fail due to DNA degradation or insufficient template. Analysis of mtDNA may be possible in such cases because human cells contain hundreds to perhaps thousands of mtDNA copies. In addition, mtDNA is circular and more resistant to exonuclease digestion. However, even though abundant on a cellular basis, mtDNA quantities may be limited. Thus, judicious use of a sample is important so that sample consumption is minimized and amplification of mtDNA template is optimized.
Another important consideration in forensic mtDNA analysis is inhibition of amplification. Hair and calcified tissue may contain PCR inhibitors, such as melanin and humic acid, respectively, at levels that could interfere with PCR and subsequent analysis. Internal positive controls (IPCs) may be used to determine whether a sample contains PCR inhibitors or insufficient template.
Real-time quantitative PCR (qPCR), a highly specific, sensitive, and reproducible method for quantifying nucleic acids, represents an attractive approach to quantifying mtDNA. Using a DNA standard, actual copy numbers of a target sequence in a sample may be determined via qPCR. Optimally, the standard contains a DNA sequence, which is different from, but associated with or linked to, the sequence to be analyzed, to eliminate the risk of contamination by the standard. Human mtDNA forensic analysis involves the non-coding hypervariable regions (HVRs) where most variation in mtDNA is found. The mtDNA coding region also contains sequence variation; therefore it is difficult to generate or identify a mtDNA standard that lacks sequence variation. Nonetheless, the present invention utilizes a unique standard and corresponding target sequence that represents a mtDNA coding sequence containing minimal variation.
The prior art discloses several qPCR assays for quantifying human mtDNA. Many such prior art assays utilize highly specific fluorogenic probes and mtDNA-specific standard curves, which enable the absolute quantification of mtDNA down to 10 copies of mtDNA or less. Other prior art assays are characterized by a high specificity for human DNA and incorporate an IPC to detect PCR inhibitors, which serves to identify those samples that may require additional purification.
The qPCR-based invention described herein combines these features as well as the ability to quantify degraded DNA, high reproducibility, and a wide dynamic range to enable quantification of low copy number samples, such as hair shafts, bone, and degraded blood, as well as high copy number samples, such as fresh blood and buccal swabs. In addition, the present invention utilizes a unique DNA standard that provides quality control and lacks topological constraint, common with plasmid-based standards, thereby allowing for greater accuracy of quantification.